HK1101085A - Method of removing adhesive microvesicles - Google Patents

Description

Method for removing adherent microbubbles

Technical Field

The present invention relates to a method for pretreating adherent microvesicles generated in a blood-derived sample, which does not affect the determination of circulating microvesicles present in the blood prior to the collection of a blood sample when measuring microvesicles, a method for correctly determining circulating microvesicles, and a reagent or kit for the determination thereof.

Background

Activated platelets release microbubbles when thrombosis occurs in atherosclerosis, disseminated intravascular coagulopathy, and such conditions. Microvesicles are known to have various functions such as a coagulation promoting effect by binding/aggregation of coagulation factors to phospholipids on the membrane surface, an activation effect on monocytes, platelets and various endothelial cells, and an adhesion promoting effect on leukocyte-platelet/leukocyte-leukocyte/endothelial cell-leukocyte. Therefore, microvesicles are considered not only as markers of platelet activation but also closely related to the progression of thrombosis and arteriosclerosis (see non-patent document 1). Therefore, one of the important diagnostic methods for predicting the disease state related to the above events is to measure the amount of microvesicles in blood. However, platelets are easily activated by physical stimuli, whereby microvesicles are released from the activated platelets. For example, due to physical shock during blood sampling or during separation of plasma or serum, contaminated platelets are activated, thereby increasing microbubbles that do not reflect the clinical condition of the blood-derived sample after blood sampling. These microbubbles increase the amount of microbubbles present in the blood-derived sample and make it difficult to accurately determine the condition of the patient. Therefore, there is a need for a method for accurately measuring microvesicles in blood before blood sampling by eliminating the influence of microvesicles generated after blood sampling.

Non-patent document 1: nomura s, int.j.hemtool, 74: 397-404, 2001

Disclosure of Invention

Problems to be solved by the invention

In order to solve the above-mentioned problems caused by the microbubbles (adhesive microbubbles) generated in the preparation of the blood-derived sample after the collection of the blood sample, the present invention has an object to provide a method for eliminating the adhesive microbubbles, a method for accurately measuring the circulating microbubbles in the blood by using the method, and a reagent for the measurement.

Means for solving the problems

From this fact, through the infiltration studies, the inventors of the present invention have found that albumin, typically BSA (bovine serum albumin), specifically interacts with microbubbles (adherent microbubbles) generated after blood sample collection to eliminate its influence in the immunoassay and to facilitate correct determination of microbubbles (circulating microbubbles) present only before blood sample collection by the immunoassay. The present invention is based on the foregoing results in full, and specifically provides the following inventions.

[1] A method for pretreating a blood-derived sample for eliminating the influence of adherent microbubbles generated in the blood-derived sample after collection of a blood sample, characterized in that albumin is added to the blood-derived sample.

[2] The method according to [1], wherein the albumin is BSA.

[3] A method for immunoassay of circulating microvesicles in a blood-derived sample, comprising the step of eliminating the influence of adherent microvesicles generated in the blood-derived sample after blood sample collection by adding albumin to the blood-derived sample, and the step of immunologically analyzing the circulating microvesicles in the blood-derived sample.

[4] The method according to [3], wherein the circulating microvesicles are immunologically analyzed by linking an antibody against the circulating microvesicles to a surface epitope specific to the microvesicles and determining the level of the antibody bound thereby.

[5] The method according to [4], wherein the surface epitope as described above is present on a glycoprotein of a circulating microvesicle.

[6] The method according to [5], wherein the surface glycoprotein as described above is GpIb-IX.

[7] The method according to [6], wherein the antibody is detected based on a label or an enzyme bound to the antibody.

[8] The method according to [7], characterized by comprising a step in which the circulating microvesicles are bound with an immobilized antibody against GpIX, followed by reacting an antibody recognizing GpIb on the microvesicles.

[9] The kit for determining the circulating microvesicles in the blood-derived sample at least comprises albumin, a marked anti-GpIb antibody and an anti-GpIX antibody.

[10] The kit according to [9], wherein the albumin is BSA.

[11] The kit according to [9] or [10], wherein the anti-GpIX antibody is coated in a plate.

Herein, the microvesicles present in the blood of a patient are referred to as "circulating microvesicles", whereas the microvesicles released on platelets activated by manual manipulation during or after blood sample collection are referred to as "adhesive microvesicles" because of their affinity to albumin.

A blood-derived sample as set forth herein refers to a sample that contains blood or a solution of one or more blood components prepared from the blood, and preferably in which platelets have been removed. Specifically, the sample may include plasma, and may include a solution obtained by diluting with physiological saline or a buffer solution, depending on the measurement of blood fractions.

The albumin refers to albumin derived from human, mammals other than human and birds, and the mammals other than human may include, for example, cows, horses, monkeys, dogs, rabbits and mice, and the birds may include chickens as a typical example. In particular, Bovine Serum Albumin (BSA) may be included as a typical example, and in addition, human serum albumin (HAS) and ovalbumin may be included as suitable examples.

For the time of adding the albumin, albumin may be added to the blood-derived sample before the assay, and the albumin may be added after the antibody is immobilized on the plate, for example, in an ELISA method. The form of addition is not particularly limited, but it is preferably added in a liquid state, and the albumin is dissolved in a physiological saline or an isotonic buffer in advance, and then may be added. The amount of albumin to be added is preferably 5% or more of the final concentration, for example, a typical final concentration is about 5 to 10%. After the addition, preferably by standing or shaking so that adherent microbubbles are adsorbed to the albumin, the treatment of the albumin is preferably carried out for one hour or more, and preferably for 4 hours or more. Thus, microbubbles released from artificially activated platelets interact with the albumin, are cleared out of the immunoassay system by the use of antibodies to microbubble membrane surface antigens, and have substantially no effect on the determination of circulating microbubbles.

Treatment with albumin does not substantially affect circulating microvesicles originally in the blood-derived sample, and the circulating microvesicles can be determined by standard immunoassays using microvesicle characteristics.

The immunization method is not particularly limited as long as it is a procedure for performing measurement and quantification of microvesicles by using an antibody against an epitope (epitope) on the surface of the membrane of the microvesicles and by quantifying the molecule based on the antigen molecule. For example, a typical ELISA method, RIA method or aggregation method.

Preferred antibodies of the invention may include antibodies against the GpIb-GPIX complex and/or glycoproteins (including individual glycoproteins, GpIb or GPIX). Other glycoproteins observed on microvesicles that are specific for the platelets include GpIa-IIa, GpIIb-IIIa, and GpIIb.

The antibody bound to the circulating microvesicles may be labeled by itself, or a labeled secondary antibody, e.g., a labeled anti-mouse immunoglobulin specifically binding to a mouse monoclonal antibody (primary antibody) may be used. Either polyclonal or monoclonal antibodies may be used, but preferably monoclonal antibodies are used. The label may be any component capable of giving a signal, such as an enzyme (peroxidase, alkaline phosphatase, etc.), a chromophore, a fluorophore (FITC, etc.), a radioisotope, a coloured particulate, a dye, a colloidal metal, etc.

Further, an embodiment of the present invention will be described in detail hereinafter by performing ELISA using, for example, BSA.

An aliquot of 10 to 150 μ l of the blood-derived sample component prepared above was added to each well of a 96-well plate which had been coated with an anti-GpIX antibody in advance and blocked with skim milk, and then, a BSA solution was added thereto to give a final concentration of 5%. Thereafter, the plate is incubated at room temperature or 37 ℃ for one hour or more, preferably 4 hours or more, in order to adsorb the newly produced adherent microbubbles onto the BSA. Subsequently, the plate is washed thoroughly with a washing solution, such as about 0.02 to 0.1% Tween 20/PBS. Then, peroxidase-labeled anti-GpIb antibody was added. The plate was thoroughly washed with the same washing solution. A substrate for the enzyme is then added to produce a color. Therefore, by using an antibody based on the membrane surface epitope GpIb-GpIX complex, it is possible to quantify the amount of circulating microvesicles in terms of the amount of colored substrate produced by the enzymatic reaction. By the above operation, the adherent microbubbles generated by the manual operation were captured by BSA and were not reacted with the antibody used in the serial immunoassay. It is thus possible to measure only the target circulating microvesicles originally present in the blood.

A kit for measuring circulating microvesicles, which contains various antibodies and reagents, etc. used for the above-described procedures, can be provided. Specifically, albumin represented by BSA or a solution thereof is included in the kit for effectively removing adhesive microbubbles generated by manual operation, an antibody recognizing the protein complex, which is typically represented by an antibody against GpIb and GpIX on the surface of a microbubble membrane (one or both of the antibodies are labeled), a second antibody and an enzyme substrate (when an enzyme is used as a label), if necessary, and the like. For example, when the antibody used for recognition is an anti-GpIb antibody that binds to biotin, peroxidase-labeled avidin may be included as the second reagent of the assay kit. Thus, a kit for measuring circulating microvesicles comprising a peroxidase substrate can be provided.

As will be apparent from the following examples, it has been found that adherent microvesicles, which are normally released from contaminated activated platelets during the blood treatment stage, can be sufficiently cleared from the immunoassay system by pretreatment with albumin, whereas only circulating microvesicles originally present in the blood can be measured.

Drawings

Figure 1 shows adsorption experiments of adherent microvesicles by BSA using artificially activated samples, samples obtained from patients with high microvesicles, and normal samples.

Best mode for carrying out the invention

The present invention will be described in more detail with reference to examples. The examples are described for illustration only and do not limit the invention.

Example 1

The method comprises the following steps:

1. preparation of false positive (activated) samples

Whole blood samples containing 3.8% citric acid, from healthy adult males with normal circulating microvesicle (PDMP: platelet derived microparticles) values, were stored in a refrigerator at 4 ℃ for 12 hours to newly generate PDMP (adhesive microvesicles). The cooled whole blood (2mL) was centrifuged at 3000rpm for 20 minutes in a desk top centrifuge, and the supernatant (600. mu.L) was collected to prepare an activated sample. The activated samples were diluted 2-fold and 4-fold with physiological saline and used for ELISA.

2. Sample preparation with high numerical values

From subjects (adult males) showing a high PDMP value, blood was sampled with EDTA/ACD (citric acid dextrose) (2mL), centrifuged at 3000rpm at room temperature for 20 minutes in a desk top centrifuge, and the supernatant (600. mu.L) was collected. Samples with high values were diluted 2-fold and 4-fold with physiological saline for ELISA.

3. Preparation of Normal samples

From healthy adult males whose PDMP values indicate normal, blood samples were prepared with EDTA/ACD, centrifuged at 3000rpm for 20 minutes in a bench top centrifuge, and the supernatant (600. mu.L) was collected for ELISA.

4.ELISA

The sandwich ELISA system was prepared in glycoproteins present on the surface of microvesicles membranes using antibodies against CD42b (GpIb) and CD42a (GpIX) (anti-CD 42b antibody: NNKY5-5, anti-CD 42a antibody: KMP-9), said CD42b and CD42a being molecules specific for platelets. A50. mu.L aliquot of the activated sample or samples with high values was added to each well of a 96-well microplate for ELISA (supplied by Corning: MaxISorb) which had been coated with anti-CD 42a antibody, followed by adding 50. mu.L of PBS, 2.5%, 5%, 7.5% or 10% BSA to each well to give a final concentration of BSA of 1.25%, 2.5%, 3.75% or 5%. The plates were incubated at room temperature for 4 hours with shaking. The plate was then washed with a wash solution (0.05% Tween20/PBS), biotinylated anti-CD 42b antibody was added, the color developed by the addition of peroxidase-labeled avidin, and the absorbance at 450nm was read with an immuno reader (Immunoreder) (FIG. 1).

Results

In the activated samples showing false positives, the decrease in the measured values depends on the concentration of BSA added to the wells in the assay, and at a final BSA concentration of 5%, the values decrease to values close to those of the normal samples. Meanwhile, a sample having a high value, which initially shows a high value, shows a constant value regardless of the concentration of BSA. From these results, it was confirmed that PDMP newly produced by the manipulation after blood sample collection can be selectively eliminated in the analysis by adding BSA at a final concentration of 5%.

Claims (11)

1. A method for pretreating a blood-derived sample for eliminating the influence of adherent microbubbles generated in the blood-derived sample after collection of a blood sample, characterized in that albumin is added to the blood-derived sample.

2. The method of claim 1, wherein albumin is BSA.

3. The method for immunoassay of circulating microvesicles in a blood-derived sample comprises the step of eliminating the influence of adhesive microvesicles generated in the blood-derived sample after blood sample collection by adding albumin into the blood-derived sample, and the step of immunologically analyzing the circulating microvesicles in the blood-derived sample.

4. The method of claim 3, wherein said circulating microvesicles are immunologically analyzed by linking an antibody against said circulating microvesicles to a surface epitope specific to said microvesicles and determining the level of antibody bound thereby.

5. The method of claim 4, wherein the surface epitope is present on a glycoprotein of a circulating microvesicle.

6. The method of claim 5, wherein the surface glycoprotein is GpIb-IX.

7. The method of claim 6, wherein the antibody is detected based on a label or enzyme bound to the antibody.

8. The method of claim 7, comprising the step of binding circulating microvesicles to an immobilized antibody against GpIX and subsequently reacting the antibody recognizing GpIb on the microvesicles.

9. The kit for determining the circulating microvesicles in the blood-derived sample at least comprises albumin, a marked anti-GpIb antibody and an anti-GpIX antibody.

10. The kit of claim 9, wherein the albumin is BSA.

11. The kit according to claim 9 or 10, wherein the anti-GpIX antibody is coated in a plate.